Mosquera2013_1874-1878

ARTICLE

Received 4 Jan 2013 | Accepted 28 Mar 2013 | Published 21 May 2013

Stimuli-responsive selection of target DNAsequences by synthetic bZIP peptidesJesus Mosquera1, Adrian Jimenez-Balsa1, Veronica I. Dodero1, M. Eugenio Vazquez1 & Jose L. Mascarenas1

One of the strategies used by nature to regulate gene expression relies on the stimuli-

controlled combination of DNA-binding proteins. This in turn determines the target-binding

site within the genome, and thereby whether a particular gene is activated or repressed. Here

we demonstrate how a designed basic region leucine zipper-based peptide can be directed

towards two different DNA sequences depending on its dimerization arrangement. While the

monomeric peptide is non-functional, a C-terminal metallo-dimer recognizes the natural

ATF/CREB-binding site (50-ATGA cg TCAT-30), and a N-terminal disulphide dimer binds

preferentially to the swapped sequence (50-TCAT cg ATGA-30). As the dimerization mode can

be efficiently controlled by appropriate external reagents, it is possible to reversibly drive the

peptide to either DNA site in response to such specific inputs. This represents the first

example of a designed molecule that can bind to more than one specific DNA sequence

depending on changes in its environment.

DOI: 10.1038/ncomms2825

1 Departamento de Quımica Organica and Centro Singular de Investigacion en Quımica Bioloxica e Materiais Moleculares (CIQUS), Universidade de Santiagode Compostela, 15782 Santiago de Compostela, Spain. Correspondence and requests for materials should be addressed to J.L.M.(email: [email protected]).

NATURE COMMUNICATIONS | 4:1874 | DOI: 10.1038/ncomms2825 | www.nature.com/naturecommunications 1

& 2013 Macmillan Publishers Limited. All rights reserved.

mailto:[email protected]

http://www.nature.com/naturecommunications

The pattern of gene expression in a multicellular organism isdetermined by the action of defined extracellular cues and/or environmental signals that ensure the production of

specific proteins only when and where needed. In most of thecases, these signals work by switching the activity of transcriptionfactors (TFs), key gene regulatory proteins that promote theexpression of selected genes by binding to relatively shortstretches of specific regions of the DNA1,2. Among the differentmechanisms used by nature to activate or inactivate TFs, thosethat affect their DNA-binding properties are particularlyprominent3–5. In this context, it is well known that many TFsare unable to bind to their DNA targets by themselves and needto form cooperative multimeric complexes with other partners.This is a clever regulatory mechanism used by nature that allowsthe combinatorial interaction with a large number of sites using arelatively small set of monomeric transcription regulators6–8. Forinstance, the basic region leucine zipper (bZIP) family of DNA-binding proteins can bind DNA as homo- or heterodimers, andin this way recognize different DNA sites depending on theregulated combination of dimerization partners9,10.

Over the last years there have been many reports on the designand preparation of short synthetic peptides capable of reprodu-cing the basic DNA recognition properties of natural TFs11–13.Particularly relevant are those based on dimeric bZIP basicregions, in which the natural C/C-terminal leucine zipper isreplaced by artificial dimerizers14–17. Some of these designs havegone even further and incorporate switchable elements that allowconditional off/on DNA binding by the application of externalstimuli such as light or metal ions18–22. Despite advances in theseand other switchable DNA binders23,24, designed systems capableof interacting with alternative DNA sites in an externallyregulated manner are unknown. Herein we prove for the firsttime the concept of ‘site switchability’ in the DNA binding ofsynthetic systems, and demonstrate how a single bZIP-basedpeptide can be directed towards two different DNA sequencesdepending on its dimerization arrangement. As the dimerizationcan be controlled by the application of selected reagents, themethodology allows remote control of the DNA site selectivity. Inaddition to providing a simplified model of the naturalmechanism of combinatorial DNA recognition, our work setthe bases for future achievements on the stimuli-controlleddifferential expression of selected genes.

The bZIP family of TFs is characterized by a C-terminal coiled-coil leucine zipper dimerization domain and a highly charged,N-terminal basic region, that mediates DNA binding25–27. Inaddition to the straightforward substitutions of the dimerizationdomain described before, the GCN4 bZIP-binding domain has

inspired the development of a variety of synthetic, sequence-selective DNA-binding peptides28–34. Interestingly, although inall naturally occurring bZIP proteins the basic region ispositioned N-terminal to the leucine zipper, Oakley andcoworkers35 showed that model ‘reverse’ bZIP peptides, inwhich the basic region of the bZIP factor GCN4 is placedC-terminal to its leucine zipper, can specifically bind toinverted DNA sites. Thus, while natural GCN4 C/C-terminaldimers specifically bind to ATF/CREB (50-ATGA(c/g)TCAT-30)or AP1 (50-ATGA(c)TCAT-30) sites, reversed N/N-terminaldimers bind to inverted 50-TCAT(y)ATGA-30 sites.Additionally, Morii et al.36 had also previously describedsynthetic N-terminal dimers of GCN4 with selectivity towardsthe swapped-binding site.

On these grounds, we envisioned that an appropriatedimerization of a GCN4 basic region through either its C- orthe N-terminal side might allow to target two different sites by thesame peptide unit. In addition, if the peptide is equipped withorthogonally responsive elements so that either dimerization canbe triggered in response to different external stimuli, we would bein position to couple site selectivity to external inputs (Fig. 1). Theresulting system would, therefore, represent a simplified model ofthe combinatorial DNA recognition in natural TFs. Reversibilitywould be an additional and highly desirable property as it mightendow site switchability.

ResultsPeptide design and synthesis. Based on earlier reports bySchepartz and co-workers17,37 on the use of metal-terpyridinecomplexes as dimerization elements for bZIP basic regions, wechose a terpyridine chelating ligand for the C-terminal end of theGCN4 basic region. This would allow promoting the dimerizationby addition of a metal ion and formation of a stable metalcomplex with two terpyridine-tagged basic regions, whileproviding for reversibility. For the N-terminal dimerization, weselected a disulphide bond, as it could be easily implemented byintroduction of a N-terminal cysteine residue. The disulphidebridge is sensitive to oxidizing or reducing conditions, and shouldbe chemically orthogonal to the metal-mediated C-terminaldimerization.

The starting point for our design is the truncated Asp226-Gln248 GCN4 basic region, which had been identified as theminimum length required for specific DNA binding14. This coresequence was extended with a cysteine residue at the C-terminus(introduced as trityl derivative) that would act as a nucleophilichandle for attachment of the terpyridine ligand. The N-terminal

Figure 1 | Representation of a stimuli-responsive dimerization strategy for specific binding to two DNA sites. A modified peptide derived from the

GCN4 TF contains orthogonal functionalities at both termini that in response to different stimuli (represented as a cube or a cylinder) result in

alternative dimerization states with different DNA-binding preferences: 50-ATGA(y)TCAT-30 for the natural C/C-terminal dimer (left), or reversed

50-TCAT(y)ATGA-30 for the non-natural N/N-terminal dimer (right).

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2825

2 NATURE COMMUNICATIONS | 4:1874 | DOI: 10.1038/ncomms2825 | www.nature.com/naturecommunications



connection was engineered by adding a Cys–Gly linker to theLys231 side chain, which was introduced as an Alloc-protectedderivative to allow its selective deprotection on the solid-phaseusing palladium catalysis. The resulting linker is geometricallyequivalent to the linker described by Morii et al.36 in their studiesof N-terminal covalent dimers of GCN4 basic regions(Supplementary Fig. S1). In order to differentiate both thiols,the N-terminal Cys was introduced protected as Cys(tButhio),which is stable to trifluoroacetic acid (TFA) but can be selectivelydeprotected using thiols, such as dithiothreitol or dithioerythritol.Assembly of the peptide chain in solid-phase, followed by astandard TFA deprotection/resin cleavage step, gave the desiredCys(tButhio)-protected basic region (1) in good yield (Fig. 2).Alkylation of the free thiol with 40-methylbromo-2,20:60,200-terpyridine, followed by treatment of the resulting peptide withdithioerythritol produced the desired thiol 3a. In addition, acontrol peptide (3b), in which the terpyridine is replaced by a40-nitroacetophenone chromophore (NAP), was also synthesized.

Monomeric peptides 3a and 3b were dimerized by treatmentwith DTNB (5,50-dithiobis-(2-nitrobenzoic acid), Ellman’sreagent) in phosphate buffer at pH 7.5. On the other hand,addition of 5 equiv. of Ni(ClO4)2 to 3a in neutral water promotesthe formation of the expected nickel-bis-terpyridine complex, asdeduced from liquid chromatography (LC)-mass spectrometry(MS) (Supplementary Fig. S5).

DNA-binding studies. The DNA-binding properties of the syn-thetic peptides were studied by electrophoretic mobility shiftassays (EMSA) under non-denaturing conditions, and usingSYBR-gold for DNA staining. As expected, while monomericpeptide 3a does not bind to short double-stranded (ds)DNAscontaining the direct ATF/CREB consensus DNA-bindingsequence (dir: 50-yATGA cg TCATy-30), addition of 5 equiv.of Ni(ClO4)2 results in a new slow-migrating band, consistentwith the formation of the specific complex between the (3a)2Nidimer and the DNA (Fig. 3a, lanes 1–3).

Importantly, the dsDNA inv, which features an invertedtarget site (50-yTCAT cg ATGAy-30), as well as a controldsDNA containing a single consensus half-site (hs: 50-yATGAcg TTCGy-30), did not lead to any new bands in theEMSA assays (Fig. 3a, lanes 4–9). These results demonstratethat the metal-promoted dimers of 3a show a clear preferencefor binding to the consensus direct site over the invertedalternative. With regard to the N-terminal disulphide dimer(3a)2SS, we observed the formation of a DNA complex with itstarget inverted DNA (inv, Fig. 3b, lanes 4–6) but, curiously,it also produces retarded bands with the other oligos dir and hs(Fig. 3b, lanes 1–3, and 7–9). These bands with the non-con-sensus dsDNAs are more diffuse than those observed with thedirect DNA, and most probably arise from a relatively loosecomplex in which one of the basic regions is interacting specifi-cally in the major groove of the consensus site while the othermakes nonspecific contacts38. Curiously, the control disulphide(3b)2SS, in which the terpyridine was replaced by aninert chromophore, did not elicit such interactions with thenon-consensus DNAs (Fig. 3c), which suggests that theterpyridine unit might be contributing to the formation of theundesired EMSA bands.

Changing the dimerization state. Despite the selectivity problemof the N-terminal disulphide dimer, peptide 3a seemed appro-priate to explore the viability of controlling site-interactions byaltering its dimerization state in the presence of DNA. Asexpected, reduction of the disulphide bond of (3a)2SS by treat-ment with the reducing agent TCEP (tris(2-carboxyethyl)pho-sphine) resulted in the formation of monomeric species andhence complete loss of DNA binding to either the direct or theinverse DNA sequence (Fig. 4, compare lanes 2 and 6 versus lanes3 and 7, respectively). Importantly, addition of Ni(ClO4)2 to thesolution obtained after the reduction step generates the C-term-inal (3a)2Ni dimer, as deduced by the appearance of the corre-sponding retarded DNA band when incubated with the dsDNA

DPAAL Lys RAR... ...KLQ AcHN Cys

226 248

TrtSHN

NH

O

tBuSS

AcHN

O

TFA, CH2Cl2, TIS, H2O

(90 : 5 : 2.5 : 2.5)

DPAAL Lys RAR... ...KLQAcHN Cys NH2

HS

HN

NH

O

tBuSS

AcHN

O

N

N

N

DPAAL Lys RAR... ...KLQAcHN Cys NH2

S

HN

NH

O

tBuSS

AcHN

OR

DTE100 mM phosphate bufferpH 8.3

231

GCN4 basic region

HS

1

2a; R = tPy2b; R = NAP

(3a)2SS(3a)2Ni

PAL-PEG-PS

Ellman's reagent,phosphate bufferpH 7.5

Ni(ClO4)2miliQ water

a) BrCH2R (R = tPy)(1:1) phosphate buffer / CH3CNpH 8.3

or

b) BrCH2R (R = NAP)(1:1) phosphate buffer / CH3CNpH 8.3

tPy =NAP =

O

NO2

3bR =NAP

3aR = tPy

Ellman′s reagent,phosphate buffer

pH 7.5(3b)2SS

R

Figure 2 | Synthesis of the modified monomeric peptides and dimerization reactions. Standard solid-phase peptide synthesis yields the fully protected

peptide containing an unprotected C-terminal Cys (1), which is modified in solution to yield the terpyridine derivative (2a) or the nitroacetophenone

(NAP) control peptide (2b). Final deprotection of the N-terminal Cys side chain gives the desired bifunctional peptides 3a and 3b, which can be dimerized

as described. The full sequence of the GCN4 basic region peptide is D226PAALKRARNTEAARRSRARKLQ248.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2825 ARTICLE




dir (Fig. 4, lane 8). This result, which confirms the viability ofswitching from the disulphide N-terminal dimer to the metal-promoted C-terminal dimer, encouraged us to further refine thedesign in order to achieve better sequence selectivities.

Optimization of the design. As previously shown, in contrast tothe result with the terpyridine containing disulphide (3a)2SS, thedimer (3b)2SS showed very low affinities for the non-consensusDNAs dir and hs, which suggest that the terpyridine group isactively contributing to the weaker, undesired binding mode,probably by providing DNA intercalating capability. Therefore,we decided to use a terpyridine unit featuring two methyl sub-stituents, which might hinder that interaction. To facilitate the

synthetic procedures, and minimize solution chemistry, the ter-pyridine group was introduced on solid-phase on the side chainof a lysine residue engineered at the C-terminal position of thepeptide, which also provided for further isolating the terpyridinefrom the DNA-binding basic region.

As shown in Fig. 5, the synthetic protocol involved removal ofthe Mtt (4-methyltrityl) protecting from the first Lys side chainunder mild acidic conditions39, and coupling of 5,500-dimethyl-[2,20:60,200-terpyridine]-40-carboxylic acid to the resulting amine,followed by introduction of the rest of the peptide sequence. Thelysine at position 231 was inserted as Lys(alloc), which allowedthe selective deprotection of its side chain by Pd catalysis afteracetylation of the N-terminal Asp226 (ref. 40). Finally, theGly-Cys linker was assembled onto the Lys side chain andacetylated to give the full terpyridine-modified peptide attachedto the resin. Standard deprotection and cleavage, and high-performance liquid chromatography (HPLC) purification affordedthe desired peptide 4, in an overall 30% yield. This peptide can bereadily dimerized upon oxidation with 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB) in a slightly basic phosphate buffer.On the other hand, addition of Ni2þ or Fe2þ salts to peptide 4 ledto the formation of the expected bis-terpyridine complexes (4)2Niand (4)2Fe, which could be isolated and identified by MS(Supplementary Figs S10 and S11).

DNA-binding studies with the peptide 4. As shown in theFig. 6a, the dimer (4)2Ni, made in situ by addition of Ni(ClO4)2 tothe peptide 4, binds the consensus direct DNA dir, but does notgive rise to retarded bands with the inverse DNA inv. A titrationusing a P32-labelled ds-oligonucleotide allowed us to calculate anapparent binding constant of E299 nM at 4 �C (SupplementaryFig. S14). More importantly, in contrast to the former disulphide(3a)2SS, the new N-terminal dimer (4)2SS displayed goodselectivity for its target sequence (DNA inv) against the non-consensus dir dsDNA (Fig. 6b), although, curiously, in order toobserve neat EMSA retarded bands we found that it is importantthat the binding mixture contains Ni2þ or Co2þ metal ions. It isprobable that coordination of the metals to either terpyridine,complex observed by MS (Supplementary Fig. S12), might pre-vent unproductive folding or nonspecific DNA binding, andhence favour the formation of the desired DNA complexes.Remarkably, addition of (NH4)2Fe(SO4)2 to (4)2SS suppresses theformation of complexes with the target DNA inv, most probablybecause of the intrinsic tendency of Fe2þ to simultaneously bind

1 2 3 4 5 6 7 98

a

1 2 3 4 5 6 7 98

1 2 3 4 5 6 7 8

b

c

dir inv hs

dir inv

dir

hs

inv

(3a)2Ni (3a)2Ni(3a)2Ni

(3a)2SS (3a)2SS(3a)2SS

(3b)2SS (3b)2SS

Figure 3 | EMSA analysis of DNA binding. (a) Lanes 1–3: dsDNA dir

(which contains the target ATF/CREB-binding site), lanes 4–6: dsDNA inv

(which contains an exchanged binding site), lanes 7–9: dsDNA hs (which

contains a consensus half-site). Lanes 2–3, 5–6 and 8–9: 600 and 1200 nM

of 3a and 5 equiv. of Ni(ClO4)2, as well as excess of TCEP to prevent the

oxidation of the thiols. (b) Lanes 1–3: dsDNA dir; lanes 4–6: dsDNA inv;

lanes 7–9: dsDNA hs. Lanes 2–3, 5–6 and 8–9: 300 and 600 nM of (3a)2SS.

(c) Lanes 1–4: dsDNA dir; lanes 5–8: dsDNA inv. Lanes 2–4 and 6–8: 400,

600, 800 nM of (3b)2SS. The protocol involved addition of peptide

solutions in 20 mM Tris-HCl, pH 7.5 to 18 mM Tris-HCl, pH 7.5, 90 mM KCl,

1.8 mM MgCl2, 1.8 mM EDTA, 9% glycerol, 0.11 mgml� 1 BSA, 2.25%

NP-40 and 100 nM of DNA (4 �C, 30 min) and loading into the gel. DNA

was detected by fluorescent staining with SYBR-gold. Oligonucleotide

sequences (binding sites underlined, only one strand shown): dir: 50-CGG

ATGA CG TCAT TTTTTTTC-30; inv: 50-CGG TCAT CG ATGA TTTTTTTC-30;

hs: 50-CGG ATGA CG TTCG TTTTTTTC-30 .

1 2 3 4 5 6 7 8

dirinv

Figure 4 | EMSA analysis of DNA binding after changing the

dimerization state. Two stock solutions of dimeric peptide (3a)2SS (3 mM)

in 20 mM Tris-HCl pH 7.5 in the presence 500 nM of either dsDNAs

inv (lanes 1–4) or dir (lanes 5–8) were successively treated at room

temperature with TCEP (40 mM), 5 min, and Ni(ClO4)2 (15mM) another

5 min. Aliquots of the different solutions, each containing over 50 nM DNA,

were mixed with the loading buffer and run under non-denaturing

conditions. Lanes 2 and 6: (3a)2SS; lane 3 and 7: same mixture after

treatment with TCEP; lanes 4 and 8: previous mixture after treatment with

Ni(ClO4)2. DNA was detected by fluorescent staining with SYBR-gold.





both terpyridines, thus forming a stable macrocyclic speciesunable to bind DNA. In the presence of 5 equiv. of Ni(ClO4)2,and using radioactive EMSA experiments, we calculated a KD ofE375 nM for the binding of (4)2SS to its consensus invertedDNA inv (Supplementary Fig. S13).

To get further insights on the performance of the C- andN-terminal dimers, we measured the binding preferences insolution, and in the presence of excess of a competitor DNA(calf-thymus), using fluorescence anisotropy titrations withrhodamine-labelled oligonucleotides (Fig. 7). Thus, a solution ofRhodamine-labelled oligonucleotide dir (Rho-dir) was incubatedwith increasing concentrations of (4)2Ni in the presence of acompetitive nonspecific calf-thymus DNA (50 mM in base pairs)at 10 �C, and the anisotropy at 585 nm was measured after eachaddition. We obtained a typical titration profile, which could befitted to a simple 1:1 binding model with an apparent KD ofE670 nM. In contrast, incubation of the non-target rhodamine-labelled Rho-inv oligonucleotide under the same conditions withincreasing concentrations of this C-terminal dimer resultedin a less pronounced titration curve with a much weakerKD of E25 mM. Using similar experiments, we found that(4)2SS displays a higher affinity for the consensus rhodamine-labelled inv oligo (KDE758 nM) than for the non-targetRho-dir oligonucleotide (KDE5.3 mM). These values are inagreement with those obtained using radioactive EMSA titrations,and further support the differential binding of the two dimers.The observation of a more intense anisotropy with the two non-target DNAs ((4)2Ni/Rho-inv and (4)2SS/Rho-dir)), than with thespecific complexes ((4)2Ni/Rho-dir and (4)2SS/Rho-inv) is con-sistent with the formation of more compact species in the case ofthe specific interactions, and larger complexes with the non-targetoligonucleotides, which therefore display slower dynamics.

Finally, circular dichroism experiments were consistent withthe behaviour observed in the EMSA experiments. Thus, incuba-tion of the N-terminal disulphide dimer (4)2SS with itstarget sequence (DNA inv) resulted in a significant increase in thenegative ellipticity at 222 nm, consistent with the a-helical foldingexpected for an specific interaction14,41. This increase is

not observed with the natural ATF/CREB sequence contained inthe dir oligo; likewise, the C-terminal dimer (4)2Ni only showedan increase in the negative ellipticity in the presence of its targetdir DNA, but not with the swapped inv DNA (see SupplementaryFig. S17).

Switching between sites. Once we confirmed that peptide 4exhibits the desired orthogonal DNA binding, we were interestedin exploring the viability of a sequence-selective recognition in asituation where both target sites are simultaneously available forbinding. Thus, a stock solution containing peptide 4 was added toa 50 nM equimolar mixture of both dsDNAs, containing thedirect and the inverse sequences dir1 and inv. Note that in thiscase we use ds-oligonucleotides of different lengths in order todistinguish the binding to either of them. As anticipated, themonomer by itself is unable to elicit retarded shifts (Fig. 8a,lane 3); addition of 1 equiv. of Ellman’s reagent (DTNB) and 5equiv. of Ni(ClO4)2 to that solution resulted and in the appear-ance of a new band consistent with the formation of a specificcomplex with the inverse DNA inv (Fig. 8a, band a in lane 4, andcontrol lane 1). Interestingly, addition of TCEP to the mixture ledto the disappearance of this complex and the formation of a newretarded band involving the oligo dir1, consistent with the DNAbinding of the in situ formed dimer (4)2Ni (Fig. 8a, band b inlane 5, and control lane 2). A new addition of DTNB dismountsthis complex and regenerates binding to the inverse DNA. Despitethe excess of reagents in the mixture, it is yet possible to induce anew switch and regenerate binding to other DNA. These resultsrepresent the first proof of concept on the viability of makingmolecules that can be induced to jump between two differentDNA sites, at will, in response to specific external stimuli.

DiscussionRecent years have witnessed important advances in the designand preparation of switchable on-off DNA binders. However,molecules that target different DNA sites in response to specificsignals, or that can switch between specific DNA sites, have not

1. 5% TFA, 1% TIS, CH2Cl2

2. HATU DIEA / DMF

Me2tPy-CO2H

FmocHN

PAL-PEG-PS

1. 20% Piperidine / DMF2. Fmoc-Lys(Mtt)-OH HBTU / HOBt DIEA / DMF

FmocHN Lys

MttNH( )4

N

N

N

FmocHN Lys

HN( )4

O Me2tPy

Me Me

2. Fmoc-aa-OH HBTU / HOBt DIEA / DMF

DPAAL Lys RAR... ...KLQ GAcHN Lys

226 248

HN

HNO

O

231

( )4

O Me2tPy

HSMe2tPy

4

3. i. Fmoc-Cys(Trt)-OH, HBTU / HOBt DIEA / DMF; ii. 20% Piperidine / DMF

23cycles

(4)2SS

Ellman's reagent100 mM phosphate bufferpH 7.5

(4)2Ni

Ni(ClO4)2miliQ water Me2tPy =

4. Ac2O, DIEA / DMF5. TFA, CH2Cl2, TIS, H2O (90 : 5 : 2.5 : 2.5)

1. Pd(OAc)2, N-methylmorpholine PPh3, Phenylsilane, CH2Cl2

1. 20% Piperidine / DMF

2. i. Fmoc-Gly-OH, HBTU / HOBt DIEA / DMF; ii. 20% Piperidine / DMF

3. i. 20% piperidine / DMFii. Ac2O, DIEA / DMF

Figure 5 | Synthesis of peptide 4 and its N and C-terminal dimers. Incorporation of the terpyridine moiety at the C-terminal lysine side chain while the

peptide is still bound to the resin. The tether on the lysine 231 is similar to that used in the peptide 1 (Gly–Cys, Fig. 2).





been developed. Inspired by the concept of combinatorial generegulation, which is particularly common for bZIP-type of TFs,we have made designed derivatives of bZIP basic regions that canbe induced to bind to different DNA sites depending on theirdimerization mode. In particular, our results show that installinga suitable terpyridine moiety at the C-terminus of the basic regionof GCN4, and a cysteine residue at its N-terminus, it is possibleto induce either a C- or N-terminal dimerization by usingappropriate triggers. The C-terminal dimerization can be inducedby metals like Niþ 2, while the addition of an oxidant, such asEllman’s reagent, promotes a disulphide dimerization through theN-terminus. The resulting dimers show a more than remarkableselectivity for their cognate-binding sites. Therefore, whereas theC-terminal metallodimer binds the natural ATF/CREB-bindingsite (50-ATGA cg TCAT-30) 440 times better than the swappedsequence (50-TCAT cg ATGA-30), the N-terminal disulphidedimer binds the inverted sequence over 10 times better than the

ATF/CREB site. The high DNA-binding selectivity of eachdimeric state allows the precise targeting of each site in mixturescontaining both oligonucleotides or in the presence of an excessof calf-thymus DNA. Importantly, our results indicate that thedimerization can be also triggered in situ, in the presence of DNA.Therefore, the peptide can be driven to either DNA target site byusing specific external signals that code its dimerization mode. Itis even possible to induce a reversible ‘hopping between sites’ in astimuli-responsive manner (Fig. 8b).

This relatively simple design shows many of the features foundin natural TFs, namely cooperativity, reversibility, high selectivityand response to specific environmental signals, and thusrepresent a starting point for the design of more complex DNAcombinatorial interaction networks and for gaining a moreprecise control on the artificial regulation of genes.

MethodsGeneral. All reagents were acquired from commercial sources: dimethyl for-mamide (DMF) and TFA were purchased from Scharlau, CH2Cl2 from Panreac,CH3CN from Merck, [2,20 :60 ,20 0-terpyridin]-40-yl-methanol was purchased fromHetCat. The rest of reagents were acquired from Sigma-Aldrich. All peptidesynthesis reagents and amino-acid derivatives were purchased from GL Biochem(Shanghai) Ltd and Novabiochem. Amino acids were purchased as protected Fmocamino acids with the standard side-chain protecting scheme, except for theorthogonally protected Fmoc-Lys(Alloc)-OH and Fmoc-Lys(Mtt)-OH.

Reactions were followed by analytical reversed phase (RP)-HPLC with anAgilent 1100 series LC/MS using an Eclipse XDB-C8 analytical columna(4.6� 150 mm, 5 mm). Electrospray Ionization Mass Spectrometry was performedwith an Agilent 1100 Series LC/MSD model in positive scan mode using directinjection of the purified peptide solution into the MS. Standard conditions foranalytical RP-HPLC consisted on an isocratic regime during the first 5 min, fol-lowed by a linear gradient from 5 to 75% of solvent B for 30 min at a flow rate of1 ml min� 1 (A: water with 0.1% TFA, B: acetonitrile with 0.1% TFA). Compoundswere detected by ultraviolet absorption at 220, 270, 304 and 330 nm. Purificationwere performed by semipreparative RP-HPLC with an Agilent 1100 series LC usinga Luna 5u C18(2) 100A (5mm, 10� 250 mm) reverse-phase column from Phe-nomenex. Matrix-assisted laser desorption/ionization mass spectrometry (MS) wasperformed with a Bruker Autoflex matrix-assisted laser desorption/time-of-flight(TOF) model in positive scan mode by direct irradiation of the matrix-absorbedpeptide. Concentrations were measured using the listed extinction coefficients.

Calf-thymus DNA was acquired from Sigma-Aldrich. Oligonucleotides werepurchased from Thermo Fisher Scientific GmbH on a 0.2 mmol scale as freeze-dried solids. After solving in H2O milliQ their concentrations were measured byultraviolet absorption at 260 nm with a BioRad SmartSpec Plus Spectrophotometer.Absorbance was measured twice and concentrations were calculated applyingLambert-Beer’s equation. The molar extinction coefficients of single-strandoligonucleotides were calculated by using the following formula42, e(260 nm)¼{(8.8� #T)þ (7.3� #C)þ (11.7� #G)þ (15.4� #A)}� 0.9 � 103 M� 1 cm� 1,where #A, #T, #C, #G stand for the number of each type of bases in the DNA

1 2 3 4 5 6 7 98 10

a

b

1 2 3 4 5 6 7 98 10

Figure 6 | Radioactive EMSA analysis of the DNA-binding properties of

peptide 4 and its dimeric derivatives. (a) and (b) Lanes 1–5: dsDNA dir

(100 nM); lanes 6–10: dsDNA inv (100 nM); a small proportion (B0.1%) of

the oligonucleotides are labelled with 32P for radioactive detection. (a)

Lanes 2–5 and lanes 7–10: 200, 600, 1,000, 1,600 nM of peptide 4 with 5

equiv. of Ni(ClO4)2, and excess of TCEP. (b) Lanes 2–5 and lanes 7–10: 100,

300, 500, 800 nM of peptide (4)2SS in the presence of 5 equiv. of

Ni(ClO4)2.

0 2 4 6 8 100

0.01

0.02

0.03

0.04

0.05Rho - inv

[(4)2Ni] (mM)

0

0.005

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[(4)2Ni] (mM)

Rho -dir

0 1 2 3 4 5 6 7 80

0.01

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0.03

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[(4)2SS] (mM)

Rho -dir

0

0.005

0.01

0.015

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0 1 2 3 4

[(4)2SS] (mM)

Rho - inv

Ani

sotr

opy

Figure 7 | Fluorescence anisotropy analysis of the DNA-binding properties of peptide 4 and its dimeric derivatives. Fluorescence anisotropy titrations

of the C- and N-terminal dimers with DNA oligonucletotides in 20 mM Tris-HCl, pH 6.5, 100 mM NaCl at 10 �C. Left: 50 nM of rhodamine-labelled Rho-dir

and Rho-inv oligonucleotides in the presence of 50mM concentration (in base pairs) of nonspecific calf-thymus DNA, with increasing concentrations of

C-terminal (4)2Ni dimer (stock solution: 400mM of 4, 600 mM Ni(ClO4)2 and 5 mM of TCEP). Right: 50 nM of rhodamine-labelled Rho-dir and Rho-inv

oligonucleotides in the presence of nonspecific calf-thymus DNA (50 mM base pairs) with increasing concentrations of N-terminal (4)2SS dimer

(stock solution: 200mM of (4)2SS, 600mM Ni(ClO4)2). Lines represent best fits to a 1:1 binding model. Rho-inv: Rho-TGGAG TCAT CG ATGA CTCGT-30;

Rho-dir: Rho-TGGAG ATGA CG TCAT CTCGT-30; excitation at 559 nm and anisotropy measured at 585 nm.





strand. The concentration of Rhodamine-labelled oligonucleotides was calculatedusing the molar extinction of rhodamine. Oligonucleotides were hybridized bymixing complementary sequences at equal molar concentration (50mM), heatingat 90 �C for 10 min and then slowly cooling the mixture to room temperatureover 1 h.

The synthesis of the terpyridine moieties and their coupling to the peptides isdetailed in the Supplementary Methods.

Electrophoretic mobility shift assay. EMSA were performed with a BioRad MiniProtean gel system, powered by an electrophoresis power supplies PowerPac Basicmodel, maximum power 150 V, frequency 50.60 Hz at 140 V (constant V). Thebinding reactions were performed over 30 min in 18 mM Tris (pH 7.0), 50 mMKCl, 1.2 mM MgCl2, 0.5 mM EDTA, 9% glycerol, 0.11 mg ml� 1 BSA and 4.2%NP� 40 at 4 �C. In the experiments analysed by fluorescent staining we used100 nM of the unlabelled dsDNAs and a total incubation volume of 20 ml. In theexperiments with 32P-labelled DNAs we used B100 pM of labelled dsDNAs and100 nM of the same unlabelled dsDNAs in a 20-ml binding mixture. Use of loweramounts of DNA gave less clear gels because of precipitation. In order to preventthe oxidation of peptides with free thiols, 2 mM of TCEP is present in the incu-bation solution.

Products were resolved by polyacrylamide gel electrophoresis using a 10% non-denaturing poliacrylamide gel and 0.5� TBE buffer, and analysed by auto-radiography (when radioactivity was used) or by staining with SyBrGold (MolecularProbes: 5 ml in 50 ml of 1� TBE) for 10 min and visualized by fluorescence.

The labelling of oligonucleotides with 32P was accomplished in the followingmanner: 1 ml of the single-strand oligonucleotide which will be labelled (10 mM),1 ml kinase buffer 10� (40 mM, Tris–HCl pH 7.5, 10 mM MgCl2, 5 mM dithio-threitol), 2 ml ATP (ATP (U-32P) (5,000 Ci mmol� 1)) and 0.5 ml of the kinase (10units ml� 1); after shaking, the mixture is heated for 1 h at 37 �C, and then quen-ched by heating for 10 min at 95–100 �C.

Peptide synthesis. C-terminal amide peptides were synthesized following stan-dard peptide protocols (Fmoc/tBu strategy) on a 0.1 mmol scale using a0.19 mmol g� 1 loading Fmoc-PAL-PEG-PS resin from Applied Biosystems, usinga PS3 automatic peptide synthesizer from Protein Tecnologies. The amino acidswere coupled in fourfold excess using HBTU (O-Benzotriazole-N,N,N0,N0-tetra-methyl-uronium-hexafluoro-phosphate) as activating agent. Each amino acid wasactivated for 30 s in DMF before being added onto the resin, and couplings wereconducted for 30 min. Deprotection of the temporal Fmoc protecting group wasperformed by treating the resin with 20% piperidine in DMF for 10 min. Manualcouplings were monitored using the TNBS (trinitrobenzene sulphonate) test43. Theresin cleavage-deprotection was accomplished by shaking the resin-bound peptidesfor 2 h (B0.025 mmol), in 3 ml of the cleavage cocktail (25 ml of H2O, 25ml oftriisopropylsilane, 50ml of CH2Cl2 and 900ml of TFA). The resin is filtered, and theTFA filtrate is added to ice-cold diethyl ether (30 ml). After 10 min, the precipitateis centrifuged and washed again with 20 ml of ice-cold ether. The solid residue isdried under argon, dissolved in CH3CN/water 1:1 (2 ml) and purified bysemipreparative RP-HPLC. The collected fractions are lyophilized and stored at� 20 �C. Acetylation of peptides was carried using mixtures of Ac2O/DMF (1:2) inthe presence of 2 equiv. of DIEA (N,N-Diisopropylethylamine).

Deprotection of Lys(Alloc) side chain. The resin-bound peptide (B0.05 mmol)was treated overnight at room temperature with a deoxygenated mixture ofmethylmorpholine (55ml, 10 equiv.), triphenylphosphine (20 mg, 1.5 equiv.),phenylsilane (62ml, 10 equiv.) and Pd(OAc)2 (3.4 mg, 0.3 equiv.) in CH2Cl2(2.7 ml). The resin was filtered and washed with DMF (2� 1.5 ml� 2 min),diethyldithiocarbamate (25 mg in 5 ml of DMF, 2� 1.5 ml� 5 min), DMF(2� 1.5 ml� 2 min) and CH2Cl2 (2� 5 ml� 2 min).

Deprotecion of Lys(Mtt) side chain. The resin-bound peptide (B0.05 mmol) wastreated twice with a mixture of TFA (250 ml), triisopropylsilane (50 ml) and CH2Cl2(4.7 ml) for 5 min. The resin was then filtered and washed with CH2Cl2(2� 5 ml� 2 min).

General procedure for the obtention of the disulphide dimers. The monomericthiol-containing peptide was dissolved in 100 mM buffer phosphate (pH 7.5) togive an approximate peptide concentration of 2 mM Ellman’s reagent44 (0.8 mM inacetonitrile, 0.4 equiv.) was added, and the resulting mixture was stirred for 20 minat room temperature. The reaction was quenched using 0.1% aqueous TFA, and thecrude purified by RP-HPLC. The collected fractions were lyophilized and stored at–20 �C, after characterization. The dimeric peptides were obtained in yields over70%.

Fluorescence anisotropy. Steady-state fluorescence anisotropy measurementswere made in a Jobin-Yvon Fluoromax-3 (DataMax 2.20), coupled to a tempera-ture controller Wavelength Electronics LFI-3751, and using a standard Hellmasemi-micro cuvette (114F-QS, 10 mm light path). Titrations were carried outat 10 �C using the following parameters: lex¼ 559 nm (bandwidth 2.0 nm),lem¼ 585 nm (bandwidth 12.0 nm), 1 s integration time, and each measure wastaken in triplicate. Titrations were made by adding increasing amounts of thedesired peptide to a solution containing a rhodamine-labelled oligonucleotide

1 2 3 4 5 6 7

a

b

a

HS Me2tPy4

Ni(ClO4)2TCEP

TC

EP

Ni(ClO4)2Ellman’s reagent

Ellm

an’s reagent

5′–ATGA(c/g)TCAT–3′

5′–TCAT(...)ATGA–3′

b

K

C C

N N

S-S

Q G K248231

Figure 8 | Dynamic target DNA selection by peptide 4. (a) Radioactive

EMSA experiments: A stock solution containing an equimolar concentration

of ds-oligonucleotides dir1 and inv (3 mM, a small proportion of the

oligonucleotides are labelled with 32P) in the presence of peptide 4 (36 mM)

was successively treated at room temperature with the oxidant (DTNB (0.5

equiv.) and Ni(ClO4)2 (5 equiv.)), the reducing agent (TCEP, 40 mM), and

again DTNB (40 mM) and TCEP (80 mM); after 2–3 min of reaction,

aliquots of the solutions resulting after each step, were mixed with the

loading buffer and run under non-denaturing conditions. Lane 1: DNA inv

(50 nM) in the presence of 300 nM of (4)2SS; lane 2: DNA dir1 (50 nM) in

the presence of 600 nM peptide 4 and Ni(ClO4)2 (5 equiv.). Lanes 3–7,

aliquots of the above reaction mixtures containing both dsDNAs (B50 nM

of each) and peptide 4. Lane 3: starting mixture; lane 4: mixture after the

first addition of DTNB (and Ni(ClO4)2 ) lane 5: mixture after the addition of

TCEP; lane 6: mixture after the second addition of DTNB; lane 7: mixture

after the last addition of TCEP. dir1: 50-CGG ATGA CG TCAT TTTTTTTCG

TTACGAAGGCC-30. (b) Cartoon representation of the switching process.





(50 nM) in 20 mM Tris–HCl pH 6.5 100 mM NaCl and 50 mM concentration(in base pairs) of nonspecific calf-thymus DNA.

Circular dichroism spectroscopy. CD measurements were made in a 2-mm cell at4 �C. Samples contained 5 mM of corresponding dsDNA (when present) and 5 mMof peptides in 10 mM phosphate buffer (pH 7.5) and 100 mM of NaCl. The CDspectra of the peptides (when measured in the presence of DNA) were calculated asthe difference between the spectrum of the peptide/DNA mixture and the mea-sured spectrum of a sample of the DNA oligonucleotide.

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AcknowledgementsWe thank the support given by the Spanish grants SAF2010-20822-C02, CTQ2012-31341, CSD2007-00006, Consolider Ingenio 2010, and the Xunta de Galicia INCITE09209 084PR, GRC2010/12, PGIDIT08CSA-047209PR. J.M. and A.J-B. thank the SpanishMinistry of Education for their PhD fellowships. We also thank Manuel Marcos from theUniversity of Vigo for his support and critical help with MS experiments.

Author contributionsJ.L.M. conceived the idea and designed the project. J.M., A.J-B., and V.D. performed theexperiments. J.L.M. and M.E.V. analysed the data and wrote the paper.

Additional informationSupplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Competing financial interests: The authors declare no competing financial interests.

Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/

How to cite this article: Mosquera, J. et al. Stimuli-responsive selection of target DNAsequences by synthetic bZIP peptides. Nat. Commun. 4:1874 doi: 10.1038/ncomms2825(2013).






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